Mammalian Target of Rapamycin Contributes to the Acquired Apoptotic Resistance of Human Mesothelioma Multicellular Spheroids

Abstract

When grown as three-dimensional structures, tumor cells can acquire an additional multicellular resistance to apoptosis that may mimic the chemoresistance found in solid tumors. We developed a multicellular spheroid model of malignant mesothelioma to investigate molecular mechanisms of acquired apoptotic resistance. We found that mesothelioma cell lines, when grown as multicellular spheroids, acquired resistance to a variety of apoptotic stimuli, including combinations of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), ribotoxic stressors, histone deacetylase, and proteasome inhibitors, that were highly effective against mesothelioma cells when grown as monolayers. Inhibitors of the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (mTOR) pathway, particularly rapamycin, blocked much of the acquired resistance of the spheroids, suggesting a key role for mTOR. Knockdown by small interference RNA of S6K, a major downstream target of mTOR, reproduced the effect of rapamycin, thereby confirming the role of mTOR and of S6K in the acquired resistance of threedimensional spheroids. Rapamycin or S6K knockdown increased TRAIL-induced caspase-8 cleavage in spheroids, suggesting initially that mTOR inhibited apoptosis by actions at the death receptor pathway; however, isolation of the apoptotic pathways by means of Bid knockdown ablated this effect showing that mTOR actually controls a step distal to Bid, probably at the level of the mitochondria. In sum, mTOR and S6K contribute to the apoptotic resistance of mesothelioma cells in three-dimensional, not in twodimensional, cultures. The three-dimensional model may reflect a more clinically relevant in vitro setting in which mTOR exhibits anti-apoptotic properties.Resistance to apoptosis is a distinguishing characteristic of all tumors (1) and underlies their resistance to therapy (2). Understanding the molecular foundations of apoptotic resistance could lead to improvements in the utility of current therapies or provide new therapies altogether. In vitro, apoptotic resistance of tumor cells is usually studied using two-dimensional monolayers. However, cells can attain increased resistance when grown in three-dimensional structures, a phenomenon referred as the acquisition of multicellular resistance (3). This acquired property of tumor cells may help explain why promising findings from in vitro studies have not been easily translated into therapy (4). Consequently, in the last decade, multicellular spheroids have become a valuable tool in the study of solid tumors by representing a threedimensional system of intermediate cellular complexity between monolayer cell cultures and tumors in vivo (5, 6).Human malignant pleural mesothelioma, an aggressive thoracic cancer that arises from the pleural mesothelium, is characterized by a profound resistance to standard anti-neoplastic therapies. At present, no curative therapy is available (7). To investigate the apoptotic resistance of mesothelioma, we are increasingly utilizing in vitro three-dimension...

Malignant pleural mesothelioma cells resist anoikis as quiescent pluricellular aggregates

“…Nevertheless, phospho-Akt has previously been described as being downregulated in MPM cells cultured under NA conditions without the occurrence of apoptosis. 29,30 In this study, we found that the ERK pathway was also rapidly downregulated in NA MPM cells. By contrast, downregulation of phospho-Akt and phospho-ERK in non-tumoral MeT-5A was associated with anoikis.…”

Spheroid-based 3D Cell Cultures Enable Personalized Therapy Testing and Drug Discovery in Head and Neck Cancer

“…Furthermore, in 3D models, cells seem to express distinct genes usually associated with drug resistance that were also found in in vivo animal studies (58). Factors, such as alteration of chromatin structure, apoptosis inhibition, cell cycle and permeability, make the MCTS a more reliable and predictable drug testing in vitro model compared to 2D monolayer models (29,37,(62)(63)(64). Radiation resistance has also been linked to spheroid models in the past.…”

Implication of necrosis-linked p53 aggregation in acquired apoptotic resistance to 5-FU in MCF-7 multicellular tumour spheroids

“…The acquired MCR has been demonstrated to be linked to a limitation of drug diffusion, to the activation of survival pathways such as NF-κB or mTOR, or to the upregulation of p27 (5)(6)(7)(8)(9). However, the molecular basis of MCR is not yet clear.…”

“…However, the 2D monolayer in vitro model does not exhibit the full range of drug resistance observed in in vivo solid tumours. For this reason, three-dimensional (3D) multicellular tumour spheroids (MTS) have been used as an in vitro model of solid tumours for drug resistance studies because they mimic the growth characteristics of in vivo tumours more closely than cancer cell line monolayers (5)(6)(7)(8)(9).…”